Accurate chromosome segregation requires the proper attachment between microtubules of the mitotic spindle and the centromere. The centromere is the site on the chromosome where the DNA-protein kinetochore complex is assembled. The PI identified CSE4 in a genetic screen for chromosome missegregation. Cse4 homologs have recently been identified in other organisms, including the human centromere protein, CENP-A. Cse4 and its homologs have no amino acid similarities in the N-terminal domains, but the C-terminal domains are greater than 60 percent identical to the histone-fold domain (HFD) of histone H3, a highly conserved structure common to all four core histone that form the nucleosome. The HFD of Cse4 most likely mediates the assembly of specialized nucleosomes that contain Cse4 instead of H3 and are specifically targeted to the centromere. The N-terminal tail of Cse4 is essential for function of the protein and exhibits genetic and physical interactions with known centromere components. The proposed research encompasses three major goals centered on the function of Cse4. The first is to determine the essential role of the N-terminus of Cse4 by identifying interacting proteins and determining when this function is required in the cell cycle. The second aim is to investigate how the Cse4 protein is targeted to the centromere through analysis of interactions with histone and non-histone proteins and with the CDEII element. The PI identified a novel essential gene, SCM3, as a dosage suppressor of most of the cse4 HFD mutants. Scm3 is structurally related to Nap1, a chromatin assembly factor that associates with histones and facilitates formation of the nucleosome. A model is proposed for targeting of Cse4 by Scm3 that will be tested in aim 3.